Decoy oligonucleotides and related methods

Abstract

In some aspects, the present disclosure relates to methods for reducing the detection of false positive events during analysis of a biological sample. In some aspects, the method comprises use of a decoy oligonucleotide that can hybridize to a probe or to a target nucleic acid. The methods herein have particular applicability in reducing the detection of false positive events and in combining hybridization and ligation reactions into a single step. Also provided are kits comprising probes for use in such methods.

1 . A method for analyzing a biological sample, comprising:
 a) contacting the biological sample with a probe or probe set and a decoy oligonucleotide in any suitable order, wherein:   the biological sample comprises a target nucleic acid comprising a target region,   the probe or probe set comprises a hybridization region, and   the decoy oligonucleotide comprises a decoy region capable of hybridizing to the hybridization region or the target region;   b) allowing the probe or probe set and the target nucleic acid to hybridize at one or more locations in the biological sample, wherein the decoy oligonucleotide reduces hybridization between the hybridization region and an off-target region in the biological sample; and   c) detecting a signal associated with the probe or probe set or a product thereof at the one or more locations in the biological sample, thereby detecting the target nucleic acid in the biological sample.   
     
     
         2 . The method of  claim 1 , wherein the decoy region has less than 98% sequence identity to the hybridization region of the probe or probe set. 
     
     
         3 . The method of  claim 2 , wherein the hybridization region of the probe or probe set has at least 99% sequence complementarity to the target region. 
     
     
         4 . The method of any of  claims 1 - 3 , wherein the decoy region has a lower sequence complementarity to the target region compared to the sequence complementarity of the hybridization region to the target region. 
     
     
         5 . The method of any of  claims 1 - 4 , wherein the decoy region has less than 98% sequence complementarity to the target region. 
     
     
         6 . The method of  claim 5 , wherein the decoy region has less than 95% sequence complementarity to the target region. 
     
     
         7 . The method of  claim 5  or  6 , wherein the decoy region has between about 80% and about 95% sequence complementarity to the target region. 
     
     
         8 . The method of any of  claims 1 - 4 , wherein the hybridization region has a higher sequence complementarity to the target region compared to the sequence complementarity of the hybridization region to the decoy region. 
     
     
         9 . The method of  claim 8 , wherein the hybridization region has at least 95% sequence complementarity to the decoy region. 
     
     
         10 . The method of  claim 8  or  9 , wherein the hybridization region has at least 99% sequence complementarity to the decoy region. 
     
     
         11 . The method of any of  claims 1 - 10 , wherein the decoy oligonucleotide is no more than about 10, no more than about 15, no more than about 20, no more than about 25, no more than about 30, no more than about 35, no more than about 40, no more than about 45, no more than about 50, no more than about 60, no more than about 70, no more than about 80, no more than about 90, or no more than about 100 nucleotides in length. 
     
     
         12 . The method of any of  claims 1 - 11 , wherein the decoy oligonucleotide is detectably labeled or not detectably labeled. 
     
     
         13 . The method of any of  claims 1 - 12 , wherein upon hybridization to the off-target region or the hybridization region, the decoy oligonucleotide does not comprise a region capable of directly or indirectly binding to a detectably labeled probe. 
     
     
         14 . The method of any of  claims 1 - 13 , wherein upon hybridization to the off-target region or the hybridization region, the decoy oligonucleotide is not ligatable with itself, within the probe set, or with another oligonucleotide. 
     
     
         15 . The method of any of  claims 1 - 14 , wherein upon hybridization to the off-target region or the hybridization region, the decoy oligonucleotide is not detectable by detectable probes configured to detect the probe or probe set or product thereof. 
     
     
         16 . The method of any of  claims 1 - 15 , wherein upon hybridization to the off-target region or the hybridization region, the decoy oligonucleotide is not capable of generating a product that is detectable by detectable probes configured to detect the probe or probe set or product thereof. 
     
     
         17 . The method of any of  claims 1 - 16 , wherein the product is a rolling circle amplification (RCA) product. 
     
     
         18 . The method of any of  claims 1 - 17 , wherein the probe or probe set is selected from the group consisting of: a probe comprising a 3′ or 5′ overhang upon hybridization to the target nucleic acid, optionally wherein the 3′ or 5′ overhang comprises one or more detectable labels and/or barcode sequences; a probe comprising a 3′ overhang and a 5′ overhang upon hybridization to the target nucleic acid, optionally wherein the 3′ overhang and the 5′ overhang each independently comprises one or more detectable labels and/or barcode sequences; a circular probe; a circularizable probe or probe set; a probe or probe set comprising a split hybridization region configured to hybridize to a splint, optionally wherein the split hybridization region comprises one or more barcode sequences; and a combination thereof. 
     
     
         19 . The method of any of  claims 1 - 18 , wherein the probe or probe set is not detectably labeled. 
     
     
         20 . The method of any of  claims 1 - 19 , wherein the probe or probe set further comprises a region capable of directly or indirectly binding to a detectably labeled probe. 
     
     
         21 . The method of any of  claims 1 - 20 , wherein upon hybridization to the target region, the probe or probe set is ligatable with itself, within the probe set, or with another oligonucleotide. 
     
     
         22 . The method of  claim 21 , wherein the probe or probe set is ligatable using the target region as template, with or without flap cleavage and with or without gap filling prior to ligation. 
     
     
         23 . The method of any of  claims 1 - 22 , wherein upon hybridization to the target region, the probe or probe set is capable of generating a product. 
     
     
         24 . The method of  claim 23 , wherein the product of the probe or probe set is a rolling circle amplification (RCA) product generated in situ in the biological sample. 
     
     
         25 . The method of any of  claims 1 - 24 , wherein the method comprises prior to the detecting in c), a step of removing a complex comprising the probe or probe set hybridized to the decoy oligonucleotide from the biological sample. 
     
     
         26 . A method for analyzing a biological sample, comprising:
 a) contacting the biological sample, a circularizable probe or probe set, and a decoy oligonucleotide with one another in any suitable order, wherein:   the biological sample comprises a target nucleic acid comprising a target region,   the circularizable probe or probe set comprises a first hybridization region and a second hybridization region which, upon hybridization to the target region, are ligatable, and   the decoy oligonucleotide comprises a decoy region capable of hybridizing to the first and/or second hybridization regions;   b) allowing the circularizable probe or probe set and the target nucleic acid to hybridize at one or more locations in the biological sample, wherein the decoy oligonucleotide reduces hybridization between the first and/or second hybridization regions and an off-target region in the biological sample;   c) circularizing the circularizable probe or probe set to generate a circular probe by ligating the first and second hybridization regions using the target region as template, with or without flap cleavage and with or without gap filling prior to ligation;   d) generating a rolling circle amplification (RCA) product of the circular probe; and   e) detecting a signal associated with the RCA product at the one or more locations, thereby detecting the target nucleic acid in the biological sample.   
     
     
         27 . The method of  claim 26 , wherein the circularizable probe or probe set is pre-hybridized to the decoy oligonucleotide. 
     
     
         28 . The method of  claim 27 , wherein the target region displaces the decoy region hybridized to the circularizable probe or probe set, thereby hybridizing the circularizable probe or probe set to the target nucleic acid. 
     
     
         29 . The method of any of  claims 26 - 28 , wherein the hybridization in step b) and the ligation in step c) are carried out under the same reaction condition, optionally wherein a ligase that performs the ligation is added prior to, during, and/or after the hybridization in step b). 
     
     
         30 . The method of  claim 29 , wherein the ligase is present in and/or added to a reaction buffer for the hybridization in step b). 
     
     
         31 . The method of any of  claims 26 - 30 , which does not comprise washing the biological sample and/or changing a reaction buffer between the hybridization in step b) and the ligation in step c). 
     
     
         32 . The method of any of  claims 26 - 31 , which does not comprise washing the biological sample and/or changing a reaction buffer between the contacting in step a) and the ligation in step c). 
     
     
         33 . The method of any of  claims 26 - 32 , which comprises prior to the circularizing in step c), a step of removing a complex comprising the circularizable probe or probe set hybridized to the decoy oligonucleotide from the biological sample. 
     
     
         34 . The method of any of  claims 26 - 32 , wherein a complex comprising the circularizable probe or probe set hybridized to the decoy oligonucleotide is not removed from the biological sample prior to the circularizing in step c). 
     
     
         35 . The method of  claim 34 , wherein in the complex, the decoy oligonucleotide comprises one or more mismatches with the circularizable probe or probe set at or near a ligation junction. 
     
     
         36 . The method of  claim 35 , wherein in step c), a circular probe of the circularizable probe or probe set hybridized to the decoy oligonucleotide is not generated. 
     
     
         37 . The method of any of  claims 26 - 36 , wherein the decoy oligonucleotide is not capable of being extended by a polymerase, optionally wherein the decoy oligonucleotide comprises an irreversible terminating group, optionally wherein the decoy oligonucleotide comprises a 3′ dideoxynucleotide. 
     
     
         38 . A method for analyzing a biological sample, comprising:
 a) contacting the biological sample, a circularizable probe or probe set, and a decoy oligonucleotide with one another in any suitable order, wherein:   the biological sample comprises a target nucleic acid comprising a target region,   the circularizable probe or probe set comprises a first hybridization region and a second hybridization region which, upon hybridization to the target region, are ligatable, and   the decoy oligonucleotide comprises a decoy region capable of hybridizing to the target region;   b) allowing the circularizable probe or probe set and the target nucleic acid to hybridize at one or more locations in the biological sample, wherein the decoy oligonucleotide reduces hybridization between the first and/or second hybridization regions and an off-target region in the biological sample;   c) circularizing the circularizable probe or probe set to generate a circular probe by ligating the first and second hybridization regions using the target region as template, with or without flap cleavage and with or without gap filling prior to ligation;   d) generating a rolling circle amplification (RCA) product of the circular probe; and   e) detecting a signal associated with the RCA product at the one or more locations, thereby detecting the target nucleic acid in the biological sample.   
     
     
         39 . The method of  claim 38 , wherein the target region and/or the off-target region is pre-hybridized to the decoy oligonucleotide. 
     
     
         40 . The method of  claim 39 , wherein the first and/or hybridization regions displace the decoy region hybridized to the target region, thereby hybridizing the circularizable probe or probe set to the target nucleic acid, and/or wherein the first and/or hybridization regions do not displace the decoy region hybridized to the off-target region. 
     
     
         41 . The method of any of  claims 38 - 40 , wherein the hybridization in step b) and the ligation in step c) are carried out under the same reaction condition, wherein a ligase that performs the ligation is added prior to, during, and/or after the hybridization in step b). 
     
     
         42 . The method of  claim 41 , wherein the ligase is present in and/or added to a reaction buffer for the hybridization in step b). 
     
     
         43 . The method of any of  claims 38 - 42 , which does not comprise washing the biological sample and/or changing a reaction buffer between the hybridization in step b) and the ligation in step c). 
     
     
         44 . The method of any of  claims 38 - 43 , which does not comprise washing the biological sample and/or changing a reaction buffer between the contacting in step a) and the ligation in step c). 
     
     
         45 . The method of any of  claims 38 - 44 , wherein a first decoy oligonucleotide hybridized to the target region is removed from the biological sample prior to the circularizing in step c), and/or wherein a second decoy oligonucleotide hybridized to the off-target region is not removed from the biological sample prior to the circularizing in step c). 
     
     
         46 . The method of  claim 45 , wherein the decoy oligonucleotide is circularizable, and the decoy oligonucleotide comprises one or more mismatches with the target region at or near a ligation junction. 
     
     
         47 . The method of  claim 45  or  46 , wherein the decoy oligonucleotide does not comprise a mismatch with the off-target region at or near a ligation junction. 
     
     
         48 . The method of any one of  claims 45 - 46 , wherein in the first complex and/or the second complex, the decoy oligonucleotide comprises a non-ligatable 3′ end and/or non-ligatable 5′ end. 
     
     
         49 . The method of any one of  claims 47 - 48 , wherein in step c), a circular probe is not generated of the decoy oligonucleotide hybridized to the target region or the off-target region. 
     
     
         50 . The method of any one of  claims 1 - 49 , wherein the decoy oligonucleotide lacks a phosphate group at the 5′ end. 
     
     
         51 . The method of any one of  claims 1 - 50 , wherein the decoy oligonucleotide comprises one or more modifications that reduce its ability to be used as a template for amplification. 
     
     
         52 . The method of any one of  claims 1 - 4  or  8 - 37 , wherein complementarity between the decoy oligonucleotide and the hybridization region in the probe or probe set is lower than complementarity between the hybridization region and the target region of the target nucleic acid. 
     
     
         53 . The method of any one of  claims 1 - 4 ,  8 - 37 , or  52 , wherein the decoy oligonucleotide has between about 80% and about 95% complementarity to the hybridization region in the probe or probe set. 
     
     
         54 . The method of any one of  claims 1 - 4 ,  8 - 37 , or  52 - 53  wherein the probe or probe set and the decoy oligonucleotide are provided as the first complex. 
     
     
         55 . The method of any one of  claims 1 - 54 , wherein the probe or probe set and the decoy oligonucleotide are provided separately. 
     
     
         56 . The method of any one of  claims 1 - 55 , wherein the probe or probe set and the decoy oligonucleotide are provided at a ratio of 1:1. 
     
     
         57 . The method any one of  claims 1 - 56 , wherein the probe or probe set and the decoy oligonucleotide are provided at a ratio higher than 1:1. 
     
     
         58 . The method of any one of  claims 55 - 57 , wherein the method further comprises allowing hybridization of the probe or probe set and the decoy oligonucleotide to form the first complex. 
     
     
         59 . The method of any one of  claims 55 - 57 , wherein the decoy oligonucleotide is no more than 10, no more than 15, no more than 20, no more than 25, or no more than 30 nucleotides in length. 
     
     
         60 . The method of any of  claims 1 - 59 , wherein the method further comprises removing the decoy probe hybridized to the first and/or second hybridization region or the target region prior to ligating the probe or probe set hybridized to the target nucleic acid. 
     
     
         61 . The method of  claim 60 , wherein the removing step comprises one or more stringency washes. 
     
     
         62 . The method of any one of  claims 1 - 61 , wherein the target nucleic acid is an mRNA. 
     
     
         63 . The method of any one of  claims 1 - 61 , wherein the target nucleic acid is a noncoding RNA. 
     
     
         64 . The method of any one of  claims 1 - 63 , wherein the target region comprises a single nucleotide of interest, an alternatively spliced region, a deletion, and/or a frameshift. 
     
     
         65 . The method of  claim 64 , wherein the single nucleotide of interest is selected from the group consisting of a single-nucleotide polymorphism (SNP), a single-nucleotide variant (SNV), a single-nucleotide substitution, a point mutation, or a single-nucleotide insertion. 
     
     
         66 . The method of  claim 65 , wherein the single nucleotide of interest is a SNP. 
     
     
         67 . The method of  claim 65 , wherein the single nucleotide of interest is a point mutation. 
     
     
         68 . The method of any of  claims 1 - 67 , wherein the target biological sample is a tissue sample, and the target region is analyzed in situ at a location in the tissue sample. 
     
     
         69 . The method of  claim 68 , wherein the tissue sample is an intact tissue sample or a non-homogenized tissue sample. 
     
     
         70 . The method of  claim 68  or  claim 69 , wherein the target nucleic acid is in a cell in the tissue sample. 
     
     
         71 . The method of  claim 70 , further comprising permeabilizing the cell before, during, or after the contacting step. 
     
     
         72 . The method of any of  claims 68 - 71 , wherein the tissue sample is a tissue section. 
     
     
         73 . The method of any of  claims 68 - 72 , wherein the tissue sample is a fixed tissue sample, a frozen tissue sample, or a fresh tissue sample. 
     
     
         74 . The method of any of  claims 26 - 73 , wherein the ligating is enzymatic ligation or chemical ligation. 
     
     
         75 . The method of any one of  claims 26 - 74 , wherein the ligating is performed using a ligase selected from the group consisting of a T4 RNA ligase 1, a T4 RNA ligase 2 or a PBCV-1 DNA ligase. 
     
     
         76 . The method of any one of  claims 26 - 75 , wherein ligating the probe or probe set results in a circularized probe. 
     
     
         77 . The method of any of  claims 26 - 76 , wherein detecting the ligated probe or probe set comprises generating an amplification product in situ, and detecting the amplification product. 
     
     
         78 . The method of  claim 77 , wherein detecting the probe or probe set, the ligated probe or probe set, and/or the amplification product comprises determining a sequence of the probe or probe set, the ligated probe or probe set, or the amplification product. 
     
     
         79 . The method of  claim 78 , wherein the sequencing comprises sequencing by hybridization, sequencing by ligation, and/or fluorescent in situ sequencing. 
     
     
         80 . The method of  claim 78  or  claim 79 , wherein a sequence in the amplification product indicative of the target region is determined. 
     
     
         81 . The method of  claim 78 , wherein the in situ hybridization comprises sequential fluorescent in situ hybridization. 
     
     
         82 . The method of any one of  claims 1 - 81 , wherein detecting of the probe or probe set, the ligated probe or probe set, and/or the amplification product comprises labeling the ligated probe or probe set and/or the amplification product with a fluorophore, an isotope, a mass tag, or a combination thereof. 
     
     
         83 . The method of any one of  claims 15 - 82 , wherein the amplification product is generated using a linear rolling circle amplification (RCA), a branched RCA, a dendritic RCA, or any combination thereof. 
     
     
         84 . The method of  claim 83 , wherein the amplification product is generated using a polymerase selected from the group consisting of Phi29 DNA polymerase, Phi29-like DNA polymerase, M2 DNA polymerase, B103 DNA polymerase, GA-1 DNA polymerase, phi-PRD1 polymerase, Vent DNA polymerase, Deep Vent DNA polymerase, Vent (exo-) DNA polymerase, KlenTaq DNA polymerase, DNA polymerase I, Klenow fragment of DNA polymerase I, DNA polymerase III, T3 DNA polymerase, T4 DNA polymerase, T5 DNA polymerase, T7 DNA polymerase, Bst polymerase, rBST DNA polymerase, N29 DNA polymerase, TopoTaq DNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, T3 RNA polymerase, and a variant or derivative thereof. 
     
     
         85 . The method of any of  claims 1 - 84 , wherein the probe or probe set, the ligated probe or probe set and/or the amplification product thereof are immobilized in the biological sample and/or crosslinked to one or more other molecules in the biological sample. 
     
     
         86 . The method of any of  claims 1 - 85 , wherein the method comprises imaging the sample to detect the probe or probe set, the ligated probe or probe set, and/or the amplification product thereof. 
     
     
         87 . The method of  claim 86 , wherein the imaging comprises detecting a signal associated the probe or probe set, the ligated probe or probe set, and/or the amplification product thereof. 
     
     
         88 . The method of  claim 87 , wherein the signal is amplified in situ in the biological sample. 
     
     
         89 . The method of  claim 88 , wherein the signal amplification in situ comprises RCA of a probe that directly or indirectly binds to the probe or probe set; hybridization chain reaction (HCR) directly or indirectly on the probe or probe set and/or the product thereof; linear oligonucleotide hybridization chain reaction (LO-HCR) directly or indirectly on the probe or probe set and/or the product thereof; primer exchange reaction (PER) directly or indirectly on the probe or probe set and/or the product thereof; assembly of branched structures directly or indirectly on the probe or probe set and/or the product thereof, hybridization of a plurality of detectable probes directly or indirectly on the probe or probe set and/or the product thereof, or any combination thereof. 
     
     
         90 . The method of any one of  claims 1 - 89  wherein the probe or probe set comprise one or more barcode sequences. 
     
     
         91 . The method of any of  claims 1 - 90 , wherein the probe or probe set comprise one or more barcode sequences that identifies a nucleic acid sequence. 
     
     
         92 . The method of  claim 91 , wherein the one or more barcode sequences identify the target region. 
     
     
         93 . The method of any one of  claims 90 - 92 , wherein the one or more barcode sequences are between about 8 and about 16 nucleotides in length. 
     
     
         94 . The method of  claim 93 , wherein the one or more barcode sequences are between about 8 and about 10 nucleotides in length. 
     
     
         95 . The method of any one of  claims 90 - 94 , wherein the method comprises detecting the one or more barcode sequences by:
 contacting the biological sample with one or more detectably-labeled probes that directly or indirectly hybridize to the one or more barcode sequences,   detecting signals associated with the one or more detectably-labeled probes, and   dehybridizing the one or more detectably-labeled probes,   optionally wherein the contacting, detecting, and dehybridizing steps are repeated with the one or more detectably-labeled probes and/or one or more other detectably-labeled probes that directly or indirectly hybridize to the one or more barcode sequences.   
     
     
         96 . A method for analyzing a biological sample, comprising:
 a) contacting the biological sample, with a complex comprising a circularizable probe and a decoy oligonucleotide, wherein:   the biological sample comprises a target nucleic acid comprising a target region,   the circularizable probe comprises a first hybridization region and a second hybridization region which, upon hybridization to the target region, are ligatable, and   the decoy oligonucleotide comprises a decoy region capable of hybridizing to the first and/or second hybridization regions, wherein the complementarity between the decoy region and the first and/or second hybridization region is lower than the complementarity between the target region and the first and/or second hybridization region, but the complementarity between the decoy region and the first and/or second hybridization region is higher than the complementarity between an off-target region and the first and/or second hybridization region;   b) allowing the circularizable probe to hybridize to the target nucleic acid at one or more locations in the biological sample, thereby displacing the decoy oligonucleotide;   c) circularizing the circularizable probe to generate a circular probe by ligating the first and second hybridization regions using the target region as template, wherein the ligating is performed under the same reaction conditions as the hybridizing in step b);   d) generating a rolling circle amplification (RCA) product of the circular probe; and   e) detecting a signal associated with the RCA product at the one or more locations, thereby detecting the target nucleic acid in the biological sample.   
     
     
         97 . A kit for analyzing a biological sample, comprising:
 (i) a probe or probe set comprising a hybridization region complementary to a target region in a target nucleic acid,   (ii) a first decoy oligonucleotide, wherein the first decoy oligonucleotide comprises a first decoy region having a first percent complementarity to a hybridization region of the probe or probe set or to the target region, and   (iii) a second decoy oligonucleotide, wherein the second decoy oligonucleotide comprises a second decoy region having a second percent complementarity to the hybridization region of the probe or probe set or to the target region.   
     
     
         98 . The kit of  claim 97 , wherein the first percent complementarity is higher than the second percent complementarity. 
     
     
         99 . The kit of  claim 97  or  98 , wherein the first decoy oligonucleotide is at a first concentration and the second decoy oligonucleotide is at a second concentration. 
     
     
         100 . The kit of  claim 99 , wherein the first concentration is lower than the second concentration.